As an example of fluorescent light decomposition for bioanalytical studies, in high throughput screening, the ability to simultaneously detect a plurality of fluorescent dyes with good wavelength discrimination enables deeper multiplexing and higher throughput. In another example using fluorescent light analysis, simultaneous detection of multiple dyes associated with cells allows simultaneous assay of cell surface antigens, organelle states, nucleic acid assay, and intercellular protein content to be detected in a single assay. Multiple wavelength detection requires detectors which can separate many bands of colors. This has commonly been done using dichroic mirror beam splitters.
U.S. Pat. No. 5,317,162 to B. Pinsky and R. Hoffman, assigned to the assignee of the present invention, describes an instrument for phase resolved fluorescent analysis. The architecture of that instrument is similar to prior art instruments which rely upon color decomposition of a beam of fluorescent light derived from a laser impinging upon a fluorescent target. Such an apparatus is described in the book Practical Flow Cytometry, by H. M. Shapiro, Third Edition (1995), p. 9. The book describes an apparatus similar to what is shown in FIG. 1. A laser beam 12 from an air cooled argon ion laser 11 is used to generate a fluorescent signal which is subsequently decomposed or decimated. The beam 12 passes through focusing elements 13, 14 and 15 to impinge upon a fluorescent substance in a flow cell 41. Fluorescent target material, such as fluorescently tagged cells or particles within a liquid stream 16 flow through the flow cell 41. Particles 43 having passed through flow cell 41 are collected in container 22. Flow is adjusted by a fluidic system 18 which provides a hydrodynamically focused flow of cells within a sheath fluid. As the target substance passes through the flow cell, the focused light beam 12 intersects the liquid stream, causing fluorescent excitation, including the scattering of light. A photodiode detector 21 is positioned to receive forwardly scattered light. The fluorescent light is typically collected at an angle which is 90° relative to the excitation access of the light beam 12. Axis 24 represents the 90° viewing axis for collection of fluorescent light. Objective lens 19 is placed across axis 24 to collect and collimate the fluorescent signal from the target substance. Fluorescent light collected by the lens 19 is formed into a beam 28 which impinges upon the dichroic mirror 25. The dichroic mirror reflects light above 640 nm and transmits the remainder as the transmitted leg 30. Reflected leg 31 is directed to the red light fluorescence detector photo multiplier tube (PMT) having a 660 nm longpass filter. Detector 32 thus registers the red light component of the collected fluorescent signal from the flow cell 41. The transmitted leg 30 impinges upon the dichroic mirror 34 which reflects light above 600 nm. The reflected leg 35 is orange light which is detected by the orange fluorescence detector PMT 33 having a 620 nm bandpass filter. The transmitted leg 36 impinges upon the dichroic mirror 38 which reflects light above 550 nm and transmits the remainder in transmitted leg 42. Reflected leg 39 is detected by the yellow fluorescence detector PMT 40 having a 575 nm bandpass filter.
The transmitted leg 42 impinges upon dichroic mirror 44 which reflects light above 500 nm. The reflected leg 46 impinges upon the green fluorescence detector PMT 47, while the transmitted leg 48 consists of essentially blue light which is directed into the orthogonal scatter detector PMT 50 with a 488 nm bandpass filter, registering blue light. In this manner, the fluorescent signal in beam 28 collected by collector lens 19 is decomposed into five colors with the amplitude of each detector being recorded simultaneously to form a spectral characteristic of the fluorescent material illuminated by the laser beam.
The flow cell 41 is typically a flat-sided quartz cuvet of square or rectangular cross-section with a flow path therethrough. Such a quartz cuvet of the prior art is described in international patent publication WO 01/27590 A2, owned by the assignee of the present invention, shown in FIG. 2.
In that international patent application, the flow cell mentioned above is described with an aspheric reflective light collector, unlike the lens 19 shown in FIG. 1. The apparatus of the international patent application mentioned above is shown in FIG. 2 where a flow cell 17 is a quartz block having a flow channel 20 where a liquid stream containing fluorescent material is directed through the cell in a stream controlled by a nozzle. The flow cell of FIG. 2 has a reflective aspheric light collector 51 collecting light scattered to a side of the flow cell opposite the side where lens 19 is situated. An aspheric reflective element 51, placed on the side of flow cell 17 opposite collector 19 serves to augment the light directed toward lens 19, or in some cases performs the function of lens 19. The reflective collector 51 is coated with a broadband reflecting material for augmenting the amount of light collected from the flow cell. The aspheric shape may be parabolic or ellipsoidal, having focal properties to match light collector 19 of FIG. 1.
The apparatus of FIG. 3 is described in U.S. Pat. No. 4,727,020 to D. Recktenwald and assigned to the assignee of the present invention. This device shows a pair of lasers 52 and 54 directing light to a flow cell 78 so that two different illumination profiles may be used to illuminate a sample. Each laser is selected for stimulating the desired fluorescent emission from target substances. A set of detectors is associated with a different color band. For example, laser 52 generates a beam 53 impinging upon the flow stream 78 and producing a fluorescent signal collected by lens 56, focused by lens 57 onto dichroic mirror 58, a beam splitter, for analysis by detectors 60 and 62. Similarly, laser 54 generates a beam 55 which impinges upon the flow which includes the particles under study in air and generates scattered fluorescent light, collected by light collector 56 and imaged by lens 57 onto dichroic mirror 64 where the beam is split between detectors 66 and 69. In summary, it is known that groups of detectors can be associated with different lasers simultaneously illuminating the same target substance.
An object of the invention was to provide an improved system for detecting fluorescent light having multiple colors emitted from a target using a greater number of detectors than has been achieved in the prior art.